The present invention relates generally to pharmaceutical compositions. In particular, the invention relates to microparticles with adsorbent surfaces, methods for preparing such microparticles, and uses thereof Additionally, the invention relates to compositions comprising biodegradable microparticles wherein biologically active agents, such as therapeutic polynucleotides, polypeptides, antigens, and adjuvants, are adsorbed on the surface of the microparticles.
Particulate carriers have been used in order to achieve controlled, parenteral delivery of therapeutic compounds. Such carriers are designed to maintain the active agent in the delivery system for an extended period of time. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) (see, e.g., U.S. Pat. No. 3,773,919), poly(lactide-co-glycolides), known as PLG (see, e.g., U.S. Pat. No. 4,767,628) and polyethylene glycol, known as PEG (see, e.g., U.S. Pat. No. 5,648,095). Polymethyl methacrylate polymers are nondegradable while PLG particles biodegrade by random nonenzymatic hydrolysis of ester bonds to lactic and glycolic acids, which are excreted along normal metabolic pathways.
For example, U.S. Pat. No. 5,648,095 describes the use of microspheres with encapsulated pharmaceuticals as drug delivery systems for nasal, oral, pulmonary and oral delivery. Slow-release formulations containing various polypeptide growth factors have also been described. See, e.g., International Publication No. WO 94/12158, U.S. Pat. No. 5,134,122 and International Publication No. WO 96/37216.
Fattal et al., Journal of Controlled Release 53:137-143 (1998) describes nanoparticles prepared from polyalkylcyanoacrylates (PACA) having adsorbed oligonucleotides.
Particulate carriers have also been used with adsorbed or entrapped antigens in attempts to elicit adequate immune responses. Such carriers present multiple copies of a selected antigen to the immune system and promote trapping and retention of antigens in local lymph nodes. The particles can be phagocytosed by macrophages and can enhance antigen presentation through cytokine release. For example, commonly owned, co-pending application Ser. No. 09/015,652, filed Jan. 29, 1998, describes the use of antigen-adsorbed and antigen-encapsulated microparticles to stimulate cell-mediated immunological responses, as well as methods of making the microparticles.
In commonly owned co-pending U.S. patent application Ser. No. 09/015,652 filed Jan. 29, 1998, for example, a method of forming microparticles is disclosed which comprises combining a polymer with an organic solvent, then adding an emulsion stabilizer, such as the surfactant polyvinyl alcohol (PVA), then evaporating the organic solvent, thereby forming microparticles. The surface of the microparticles comprises the polymer and the stabilizer. Macromolecules such as DNA, polypeptides, and antigens may then be adsorbed on those surfaces.
U.S. Pat. Nos. 5,814,482 and 6,015,686 disclose Eukaryotic Layered Vector Initiation Systems (ELVIS vectors), particularly those derived and constructed from alphavirus genomes (such as Sindbis virus), for use in stimulating an immune response to an antigen, in methods of inhibiting pathogenic agents, and in delivery of heterologous nucleotide sequences to eukaryotic cells and animals, among others.
Commonly owned International patent application PCT/US99/17308 and co-pending U.S. patent application Ser. No. 09/715,902 disclose methods of making microparticles having adsorbed macromolecules, such as a pharmaceutical, a polynucleotide, a polypeptide, a protein, a hormone, an enzyme, a transcription or translation mediator, an intermediate in a metabolic pathway, an immunomodulator, an antigen, an adjuvant, or combinations thereof, and the like. The microparticles comprise, for example, a polymer such as a poly(alpha-hydroxy acid) (e.g., PLG), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a polyanhydride, and the like, and are formed using, for example, cationic, anionic or nonionic detergents.
While antigen-adsorbed PLG microparticles offer significant advantages over other more toxic systems, adsorption of biologically active agents to the microparticle surface can nonetheless be improved. For example, it is often difficult or impossible to adsorb charged or bulky biologically active agents, such as polynucleotides, large polypeptides, and the like, to the microparticle surface. Thus, there is a continued need for flexible delivery systems for such agents and, particularly for drugs that are highly sensitive and difficult to formulate.
The present inventors have found that adsorption of macromolecules to microparticles can be improved by ensuring that detergent is made available for forming a complex with the macromolecules at the time of adsorption. This availability can be accomplished, for example, by separately providing a quantity of detergent at the time of macromolecule adsorption or by ensuring that the process for producing the microparticles results in a product containing a substantial amount of unbound detergent. This provision is to be contrasted with prior art techniques, where microparticles are thoroughly washed to remove residual detergent prior to macromolecule adsorption. For instance, in the Examples found in PCT/US99/17308 above, the microparticles are washed multiple times with water (i.e., they are washed with water four times by centrifugation) prior to exposure to the macromolecule of interest. Such washing steps remove essentially all unbound detergent, resulting in a final product in which greater than 99% of the remaining detergent is bound to the particles.
Thus, according to a first aspect of the invention, a microparticle composition is provided which comprises: (1) microparticles, which further comprise a polymer and a first detergent portion that is bound to the polymer; and (2) a complex of a biologically active macromolecule with a second detergent portion, which complex is adsorbed on the surface of the microparticles. The first detergent portion and the second detergent portion can comprise the same detergent or different detergents.
Preferred biologically active macromolecules are selected from the group consisting of a polypeptide, a polynucleotide, a polynucleoside, an antigen, a pharmaceutical, a hormone, an enzyme, a transcription or translation mediator, an intermediate in a metabolic pathway, an immunomodulator, and an adjuvant. Preferred polymers are poly(xcex1-hydroxy acids), more preferably those selected from the group consisting of poly(L-lactide), poly(D,L-lactide) and poly(D,L-lactide-co-glycolide). More preferred are poly(D,L-lactide-co-glycolide) polymers. Preferred poly(D,L-lactide-co-glycolide) polymers are those having a lactide/glycolide molar ratio ranging from 30:70 to 70:30, more preferably 40:60 to 60:40, and having a molecular weight ranging from 10,000 to 100,000 Daltons, more preferably from 30,000 Daltons to 70,000 Daltons. More preferred biologically active macromolecules include bacterial and viral antigens (e.g., HIV antigens such as gp120, gp140, p24gag and p55gag, meningitis B antigens, streptococcus B antigens, and Influenza A hemagglutinin antigens) and polynucleotides that encode for antigens. The biologically active macromolecule can be, for example, in the form of a plasmid, an ELVIS vector, or an RNA vector construct. A particularly preferred biologically active macromolecule is pCMV-p55gag.
In some embodiments, the microparticle composition is provided with a further biologically active macromolecule, which may be bound or unbound, and may even be entrapped within the polymer. For example, the microparticle composition may be provided with an adjuvant, particularly a Th1 stimulating adjuvant. Preferred adjuvants include CpG oligonucleotides, LTK63, LTR72, MPL and aluminum salts, including aluminum phosphate.
In some embodiments, the first detergent portion and the second detergent portion comprise the same detergent. Preferred detergents for this purpose are cationic detergents, for example, CTAB. In such embodiments, the first detergent portion (which is bound to the polymer) preferably comprises about 5-95% of the total detergent in the composition, more preferably about 10-90%, even more preferably about 10-60%, and most preferably about 25-40%.
In other embodiments, the first detergent portion and the second detergent portion comprise different detergents. For example, the first detergent portion can comprise a nonionic detergent (e.g., PVA) and the second detergent portion can comprise a cationic detergent (e.g., CTAB).
According to another aspect of the presenting invention, a pharmaceutically acceptable excipient is added to the above microparticle compositions.
Another aspect of the invention is directed to the delivery of a macromolecule to a vertebrate subject, which comprises administering to a vertebrate subject the microparticle composition above.
In an additional aspect, the invention is directed to a method for eliciting a cellular and/or humoral immune response in a vertebrate subject, which comprises administering to a vertebrate subject a therapeutically effective amount of a microparticle composition as described above.
Another aspect of the invention is directed to a method of immunization, which comprises administering to a vertebrate subject a therapeutically effective amount of the microparticle composition above.
In other aspects of the invention, the above microparticle compositions are used in the diagnosis of diseases, in the treatment of diseases, in vaccines, and/or in raising an immune response.
Still other aspects of the invention are directed to methods of producing microparticle compositions. In general, these methods comprise: (a) forming an emulsion comprising (i) a polymer selected from the group consisting of a poly(xcex1-hydroxy acid), a polyhydroxy butyric acid, a poly caprolactone, a polyorthoester, a polyanhydride, and a polycyanoacrylate, (ii) an organic solvent, (iii) a detergent and (iv) water; followed by (b) removal of the organic solvent. About 10-90% of the total detergent in the resulting composition is preferably bound to microparticles in this embodiment, more preferably about 10-60%, and most preferably about 25-40%. In general, these microparticle compositions are subsequently incubated with a biologically active macromolecule, such as those discussed above, to produce a biologically active composition.
Preferably the emulsion is a water-in-oil-in-water emulsion that is formed by a process comprising: (a) emulsifying an organic phase comprising the polymer and the organic solvent with a first aqueous phase comprising water to form a water-in-oil emulsion; and (b) emulsifying a second aqueous phase comprising the cationic detergent and water with the emulsion formed in step (a) to form a water-in-oil-in-water emulsion.
In some preferred embodiments, the detergent is a cationic detergent, which is provided in the emulsion at a weight to weight detergent to polymer ratio of from about 0.05:1 to about 0.5:1. In these embodiments, the method preferably further comprises cross-flow filtration of the particles after the solvent removal step. In a specific embodiment, the polymer is poly(D,L-lactide-co-glycolide), the cationic detergent is CTAB, and the cationic detergent is provided in the emulsion at a weight to weight detergent to polymer ratio of from about 0.1:1 to about 0.5:1.
In other preferred embodiments, the detergent is a cationic detergent that is provided in the emulsion at a weight to weight detergent to polymer ratio of from about 0.001:1 to about 0.05:1. At these lower levels, there is typically no need for a filtration or washing step to remove excess detergent. In a specific embodiment, the cationic detergent is CTAB, the polymer is poly(D,L-lactide-co-glycolide), the cationic detergent is provided in the emulsion at a weight to weight detergent to polymer ratio of from about 0.002:1 to about 0.04:1, and the microparticles are not subjected to a step to remove excess CTAB from the composition.
Still other aspects of the invention are directed to methods of producing microparticle compositions, which methods comprise: (1) providing a microparticle in an emulsification process, which microparticle comprises a polymer and a first detergent portion that is bound to the microparticle; and (2) adsorbing a complex of a biologically active macromolecule and a second detergent portion on the surface of the microparticle. The first detergent portion and the second detergent portion can comprise the same detergent or different detergents. The polymer is preferably selected from the group consisting of a poly(xcex1-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a polyanhydride, and a polycyanoacrylate.
In some embodiments, the first and second detergent portions comprise the same detergent. The detergent is preferably a cationic detergent, for example, CTAB. In these embodiments, about 10-90%, more preferably about 10-60%, and most preferably about 25-40% of the total detergent in the microparticle composition is in the form of the first detergent portion that is bound to the microparticles. Typically, all of the detergent is added during the course of the emulsification process.
In other embodiments, the first detergent portion comprises a first detergent and the second detergent portion comprises a second detergent differing from the first detergent. Typically, the first detergent is added in the course of the emulsification process and the second detergent is added subsequent to the emulsification process, preferably concurrently with the biologically active macromolecule. Preferably the first detergent portion comprises a nonionic detergent, such as PVA, and the second detergent portion comprises a cationic detergent, such as CTAB.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.